Method for high-throughput screening of transgenic plants

ABSTRACT

The invention relates to a method for quantifying levels of expression and/or quantifying copy number of a heterologous polynucleotide in a transgenic plant using quantitative or real-time polymerase chain reaction (QPCR or real-time PCR), wherein the real-time PCR is performed using a primer set specific to a heterologous terminator sequence operably linked to the heterologous polynucleotide.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/514,052, filed Aug. 2, 2011, the entire content of which is hereinincorporated by reference.

FIELD OF INVENTION

The current invention relates to plant genetic engineering. It relatesto methods and compositions for screening transgenic plants for thepresence and expression of transgenes.

BACKGROUND

Recent advances in plant genetic engineering have opened new doors toengineer plants to have improved characteristics or traits. Thesetransgenic plants characteristically have recombinant DNA constructs intheir genome that have a transgene, operably linked to multipleregulatory regions that allow accurate expression of the transgene. Afew examples of regulatory elements that help regulate gene expressionin transgenic plants are promoters, introns, terminators, enhancers andsilencers.

Plant genetic engineering has advanced to introducing multiple traitsinto commercially important plants, also known as gene stacking. This isaccomplished by multigene transformation, where multiple genes aretransferred to create a transgenic plant that might express a complexphenotype, or multiple phenotypes.

Regulatory sequences located downstream of coding regions containsignals required for transcription termination and 3′ mRNA processing,and are called terminator sequences. The terminator sequences play a keyrole in mRNA processing, localization, stability and translation(Proudfoot, N, (2004) Curr Opin Cell Biol 16:272-278; Gilmartin, G. M.(2005) Genes Dev. 19:2517-2521. The 3′ regulatory sequences contained interminator sequences can affect the level of expression of a gene(Ingelbrecht et al. (1989) Plant Cell 1:671-680).

One of the challenges of plant genetic engineering is the molecularcharacterization of the transgenic plants to detect and measure the copynumber and the expression of the transgene in the transgenic plant. Thetransgenic DNA is randomly inserted into the plant genome that canresult in gene silencing in transgenic plants with multiple transgenecopies integrated into one or more chromosomal locations. Estimation oftransgene copy number is thus a vital step of molecular characterizationof transgenic plants. Techniques such as southern blotting, comparativegenomic hybridization, fluorescence in situ hybridization and PCR usinggene-specific primers have been used to measure the copy number of thetransgene (Yang et al, Plant Cell Rep (2005) 23:759-763). Techniquessuch as Northern blotting and reverse transcriptase PCR usinggene-specific primers are used to quantify expression of the transgene.These techniques can be tedious and prone to errors (Toplak et al. 2004;Plant Molecular Biology Reporter 22: 237-250).

Using quantitative or real-time PCR for assaying transgene expression orcopy number in transgenic plants has drawbacks, as it can be costly whendone with gene specific primers and probes for each different transgene.Moreover, efficiency of each primer set might be different, which wouldhinder assays for transgene copy number and expression analysis in ahigh-throughput fashion. If the transgenic plant has an endogenous copyof the gene besides the introduced copy, measuring the expressionspecifically from the transgene can be difficult.

SUMMARY

The present invention relates to the method for quantifying levels ofexpression or quantifying copy number of a heterologous polynucleotidein a transgenic plant using quantitative or real-time polymerase chainreaction (QPCR or real-time PCR), wherein the real-time PCR is doneusing a primer set specific to a heterologous terminator sequenceoperably linked to the heterologous polynucleotide. The method describedin the current invention can be used for assaying levels of expressionor copy number of a heterologous polynucleotide in transgenic plants, ina high-throughput manner.

One embodiment of this invention is a method of quantifying the level ofexpression of a heterologous polynucleotide in a transgenic plant orplant cell, the method comprising the steps of: (a) isolating nucleicacids from a transgenic plant or plant cell, wherein the transgenicplant or plant cell comprises a heterologous polynucleotide operablylinked to a heterologous terminator sequence; and (b) quantifying thelevel of expression of the heterologous polynucleotide by real-timereverse transcriptase polymerase chain reaction using a forward primerand a reverse primer, wherein the forward primer and the reverse primerhybridize to the heterologous terminator sequence or the complementthereof. In another embodiment, the quantification of level ofexpression of the heterologous polynucleotide is done by quantitative orreal-time reverse transcriptase PCR using a probe that hybridizes to theheterologous terminator sequence or the complement thereof.

Another embodiment of the present invention is a method of measuring thecopy number of a heterologous polynucleotide in a transgenic plant orplant cell, the method comprising the steps of: (a) isolating nucleicacids from a transgenic plant or plant cell, wherein the transgenicplant or plant cell comprises a heterologous polynucleotide operablylinked to a heterologous terminator sequence; and (b) quantifying thecopy number of the heterologous polynucleotide by real-time polymerasechain reaction using a forward primer and a reverse primer, wherein theforward primer and the reverse primer hybridize to the heterologousterminator sequence or the complement thereof. In another embodiment,the quantification of copy number of the heterologous polynucleotide isdone by quantitative real-time PCR using a probe that hybridizes to theheterologous terminator sequence or the complement thereof.

Another embodiment of this invention is a method of quantifying thelevel of expression of at least two heterologous polynucleotides presentin at least two transgenic plants or plant cells, the method comprisingthe steps of: (a) isolating nucleic acids from at least two transgenicplants or plant cells, wherein a first transgenic plant or plant cellcomprises a first heterologous polynucleotide operably linked to aheterologous terminator sequence, and wherein a second transgenic plantor plant cell comprises a second heterologous polynucleotide operablylinked to the heterologous terminator sequence; (b) optionally,isolating nucleic acids from additional transgenic plants or plantcells, wherein each of the additional transgenic plants or plant cellscomprises an additional heterologous polynucleotide operably linked tothe heterologous terminator sequence; and (c) quantifying the level ofexpression of the first heterologous polynucleotide, the secondheterologous polynucleotide and optional additional heterologouspolynucleotides by real-time reverse transcriptase polymerase chainreaction using a forward primer and a reverse primer, wherein theforward primer and the reverse primer hybridize to the heterologousterminator sequence or the complement thereof. In one embodiment, theisolation of nucleic acids from additional transgenic plants or plantcells of step (b) and quantification of level of expression of optionaladditional heterologous polynucleotides of step (c) is done for at leastone hundred additional transgenic plant or plant cells. In oneembodiment the copy number of the at least two heterologouspolynucleotides present in the at least two transgenic plants or plantcells, wherein each heterologous polynucleotide is operably linked tothe heterologous terminator, is quantified using this method.

In another embodiment of any of the methods, the heterologous terminatorsequence comprises a SB-GKAF terminator sequence. In another embodiment,the sequence of the heterologous terminator sequence comprises SEQ IDNO:1. In another embodiment, the forward primer, the reverse primer andthe probe hybridize to SEQ ID NO:1 or the complement thereof. In anotherembodiment, the heterologous terminator sequence comprises the SB-GKAFterminator sequence, and the probe hybridizes to the region of theSB-GKAF terminator sequence bounded by the forward primer and thereverse primer. In another embodiment, the heterologous terminatorsequence comprises SEQ ID NO:1, the forward primer comprises SEQ IDNO:2, the reverse primer comprises SEQ ID NO:3 and the probe comprisesSEQ ID NO:4. In another embodiment, the heterologous terminator sequencecomprises SEQ ID NO:1, the forward primer comprises SEQ ID NO:5, thereverse primer comprises SEQ ID NO:6 and the probe comprises SEQ IDNO:7.

In another embodiment of any of the methods, the transgenic plant orplant cell is a maize plant or plant cell.

BRIEF DESCRIPTION OF DRAWINGS AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

FIG. 1 is a graph showing the efficiency curve of the SBTerm2primer/probe set.

FIG. 2 is a graph showing the amplification plot of the SBTerm2primer/probe set.

FIG. 3 is a schematic representation showing modes of assaying differenttransgenes using gene-specific or SB-GKAF terminator-specific primers.Each construct has a unique Gene of Interest (“GOI”), but uses the sameterminator. Each construct has a unique inner GOI primer set but usesthe same SB-GKAF primer set.

FIG. 4 is a graph showing a QPCR CT comparison using GUS and SB-GKAFterminator primer sets run on the same transgenic samples. GUS CTs areon the Y axis and SB-GKAF CTs are on the X axis.

SEQ ID NO:1 is the sequence of the SB-GKAF terminator.

SEQ ID NO:2 is the sequence of the forward primer, SBTerm2F, used forQPCR. SBTerm2F corresponds to nucleotides 33-50 of SEQ ID NO:1.

SEQ ID NO:3 is the sequence of the reverse primer, SBTerm2R, used forQPCR. SBTerm2R corresponds to the reverse complement of nucleotides91-110 of SEQ ID NO:1.

SEQ ID NO:4 is the sequence of the SBTerm2 probe used for QPCR. TheSBTerm2 probe corresponds to nucleotides 53-72 of SEQ ID NO:1.

SEQ ID NO:5 is the sequence of the forward primer, SBTerm1F, used forQPCR. SBTerm1F corresponds to nucleotides 67-91 of SEQ ID NO:1.

SEQ ID NO:6 is the sequence of the reverse primer, SBTerm1R, used forQPCR. SBTerm1R corresponds to the reverse complement of nucleotides114-135 of SEQ ID NO:1.

SEQ ID NO:7 is the sequence of the SBTerm1 probe used for QPCR. TheSBTerm1 probe corresponds to nucleotides 94-110 of SEQ ID NO:1.

SEQ ID NO:8 is the sequence of the GUS forward primer, GUS-1482-F, usedfor QPCR.

SEQ ID NO:9 is the sequence of the GUS reverse primer, GUS-1553-R, usedfor QPCR.

SEQ ID NO:10 is the sequence of the GUS probe, GUS-1509-probe, used forQPCR.

SEQ ID NO:11 is the sequence of the eIF4g forward primer, eIF4g-F, usedfor QPCR.

SEQ ID NO:12 is the sequence of the eIF4g reverse primer, eIF4g-R, usedfor QPCR.

SEQ ID NO:13 is the sequence of the eIF4g probe, eIF4g-probe, used forQPCR.

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

The terms “monocot” and “monocotyledonous plant” are usedinterchangeably herein. A monocot of the current invention includes theGramineae.

The terms “dicot” and “dicotyledonous plant” are used interchangeablyherein. A dicot of the current invention includes the followingfamilies: Brassicaceae, Leguminosae, and Solanaceae.

The terms “full complement” and “full-length complement” are usedinterchangeably herein, and refer to a complement of a given nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

“Transgenic” refers to any cell, cell line, callus, tissue, plant partor plant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those initial transgenic events as well as those created bysexual crosses or asexual propagation from the initial transgenic event.The term “transgenic” as used herein does not encompass the alterationof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. For example, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct.

The commercial development of genetically improved germplasm has alsoadvanced to the stage of introducing multiple traits into crop plants,often referred to as a gene stacking approach. In this approach,multiple genes conferring different characteristics of interest can beintroduced into a plant. Gene stacking can be accomplished by many meansincluding but not limited to co-transformation, retransformation, andcrossing lines with different transgenes.

“Transgenic plant” also includes reference to plants which comprise morethan one heterologous polynucleotide within their genome. Eachheterologous polynucleotide may confer a different trait to thetransgenic plant.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,plant propagules, seeds and plant cells and progeny of same. Plant cellsinclude, without limitation, cells from seeds, suspension cultures,embryos, meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores.

“Propagule” includes all products of meiosis and mitosis able topropagate a new plant, including but not limited to, seeds, spores andparts of a plant that serve as a means of vegetative reproduction, suchas corms, tubers, offsets, or runners. Propagule also includes graftswhere one portion of a plant is grafted to another portion of adifferent plant (even one of a different species) to create a livingorganism. Propagule also includes all plants and seeds produced bycloning or by bringing together meiotic products, or allowing meioticproducts to come together to form an embryo or fertilized egg (naturallyor with human intervention).

“Progeny” comprises any subsequent generation of a plant.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably to refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenylate or deoxyadenylate (forRNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G”for guanylate or deoxyguanylate, “U” for uridylate, “T” fordeoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C orT), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” forany nucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from anmRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Coding region” refers to the portion of a messenger RNA (or thecorresponding portion of another nucleic acid molecule such as a DNAmolecule) which encodes a protein or polypeptide. “Non-coding region”refers to all portions of a messenger RNA or other nucleic acid moleculethat are not a coding region, including but not limited to, for example,the promoter region, 5′ untranslated region (“UTR”), 3′ UTR, intron andterminator. The terms “coding region” and “coding sequence” are usedinterchangeably herein. The terms “non-coding region” and “non-codingsequence” are used interchangeably herein.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from acDNA library and therefore is a sequence which has been transcribed. AnEST is typically obtained by a single sequencing pass of a cDNA insert.The sequence of an entire cDNA insert is termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence is a sequence assembled from twoor more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein is termed a “Complete Gene Sequence”(“CGS”) and can be derived from an FIS or a contig.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product has been removed.

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature. Theterms “recombinant DNA construct” and “recombinant construct” are usedinterchangeably herein.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Overexpression” refers to the production of a gene product intransgenic organisms that exceeds levels of production in a nullsegregating (or non-transgenic) organism from the same experiment.

“Phenotype” means the detectable characteristics of a cell or organism.

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

The term “crossed” or “cross” means the fusion of gametes viapollination to produce progeny (e.g., cells, seeds or plants). The termencompasses both sexual crosses (the pollination of one plant byanother) and selfing (self-pollination, e.g., when the pollen and ovuleare from the same plant). The term “crossing” refers to the act offusing gametes via pollination to produce progeny.

A “favorable allele” is the allele at a particular locus that confers,or contributes to, a desirable phenotype, e.g., increased cell walldigestibility, or alternatively, is an allele that allows theidentification of plants with decreased cell wall digestibility that canbe removed from a breeding program or planting (“counterselection”). Afavorable allele of a marker is a marker allele that segregates with thefavorable phenotype, or alternatively, segregates with the unfavorableplant phenotype, therefore providing the benefit of identifying plants.

The term “introduced” means providing a nucleic acid (e.g., expressionconstruct) or protein into a cell. Introduced includes reference to theincorporation of a nucleic acid into a eukaryotic or prokaryotic cellwhere the nucleic acid may be incorporated into the genome of the cell,and includes reference to the transient provision of a nucleic acid orprotein to the cell. Introduced includes reference to stable ortransient transformation methods, as well as sexually crossing. Thus,“introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct/expression construct) into a cell, means“transfection” or “transformation” or “transduction” and includesreference to the incorporation of a nucleic acid fragment into aeukaryotic or prokaryotic cell where the nucleic acid fragment may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

Regulatory Sequences:

A recombinant DNA construct (including a suppression DNA construct) ofthe present invention may comprise at least one regulatory sequence.

“Regulatory sequences” or “regulatory elements” are used interchangeablyand refer to nucleotide sequences located upstream (5′ non-codingsequences), within, or downstream (3′ non-coding sequences) of a codingsequence, and which influence the transcription, RNA processing orstability, or translation of the associated coding sequence. Regulatorysequences may include, but are not limited to, promoters, translationleader sequences, introns, and polyadenylation recognition sequences.The terms “regulatory sequence” and “regulatory element” are usedinterchangeably herein.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably to refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”.

High level, constitutive expression of the candidate gene under controlof the 35S or UBI promoter may have pleiotropic effects, althoughcandidate gene efficacy may be estimated when driven by a constitutivepromoter. Use of tissue-specific and/or stress-specific promoters mayeliminate undesirable effects but retain the ability to enhance droughttolerance. This effect has been observed in Arabidopsis (Kasuga et al.(1999) Nature Biotechnol. 17:287-91).

Suitable constitutive promoters for use in a plant host cell include,but are not limited to, the core promoter of the Rsyn7 promoter andother constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812(1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, but are not limited to, forexample, those discussed in U.S. Pat. Nos. 5,608,149; 5,608,144;5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and6,177,611.

A tissue-specific or developmentally regulated promoter is a DNAsequence which regulates the expression of a DNA sequence selectively inthe cells/tissues of a plant critical to tassel development, seed set,or both, and limits the expression of such a DNA sequence to the periodof tassel development or seed maturation in the plant. Any identifiablepromoter may be used in the methods of the present invention whichcauses the desired temporal and spatial expression.

Promoters which are seed or embryo-specific and may be useful in theinvention include, but are not limited to, soybean Kunitz trypsininhibitor (Kti3, Jofuku and Goldberg, Plant Cell 1:1079-1093 (1989)),patatin (potato tubers) (Rocha-Sosa, M., et al. (1989) EMBO J. 8:23-29),convicilin, vicilin, and legumin (pea cotyledons) (Rerie, W. G., et al.(1991) Mol. Gen. Genet. 259:149-157; Newbigin, E. J., et al. (1990)Planta 180:461-470; Higgins, T. J. V., et al. (1988) Plant. Mol. Biol.11:683-695), zein (maize endosperm) (Schemthaner, J. P., et al. (1988)EMBO J. 7:1249-1255), phaseolin (bean cotyledon) (Segupta-Gopalan, C.,et al. (1985) Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324),phytohemagglutinin (bean cotyledon) (Voelker, T. et al. (1987) EMBO J.6:3571-3577), B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L,et al. (1988) EMBO J. 7:297-302), glutelin (rice endosperm), hordein(barley endosperm) (Marris, C., et al. (1988) Plant Mol. Biol.10:359-366), glutenin and gliadin (wheat endosperm) (Colot, V., et al.(1987) EMBO J. 6:3559-3564), and sporamin (sweet potato tuberous root)(Hattori, T., et al. (1990) Plant Mol. Biol. 14:595-604). Promoters ofseed-specific genes operably linked to heterologous coding regions inchimeric gene constructions maintain their temporal and spatialexpression pattern in transgenic plants. Such examples include, but arenot limited to, Arabidopsis thaliana 2S seed storage protein genepromoter to express enkephalin peptides in Arabidopsis and Brassicanapus seeds (Vanderkerckhove et al., Bio/Technology 7:L929-932 (1989)),bean lectin and bean beta-phaseolin promoters to express luciferase(Riggs et al., Plant Sci. 63:47-57 (1989)), and wheat glutenin promotersto express chloramphenicol acetyl transferase (Colot et al., EMBO J6:3559-3564 (1987)).

Inducible promoters selectively express an operably linked DNA sequencein response to the presence of an endogenous or exogenous stimulus, forexample by chemical compounds (chemical inducers) or in response toenvironmental, hormonal, chemical, and/or developmental signals.Inducible or regulated promoters include, but are not limited to, forexample, promoters regulated by light, heat, stress, flooding ordrought, phytohormones, wounding, or chemicals such as ethanol,jasmonate, salicylic acid, or safeners.

“Enhancer sequences” refer to the sequences that can increase geneexpression. These sequences can be located upstream, within introns ordownstream of the transcribed region. The transcribed region iscomprised of the exons and the intervening introns, from the promoter tothe transcription termination region. The enhancement of gene expressioncan be through various mechanisms which include, but are not limited to,increasing transcriptional efficiency, stabilization of mature mRNA andtranslational enhancement.

Recombinant DNA constructs of the present invention may also includeother regulatory sequences, including but not limited to, translationleader sequences, introns, and polyadenylation recognition sequences.

An “intron” is an intervening sequence in a gene that is transcribedinto RNA and then excised in the process of generating the mature mRNA.The term is also used for the excised RNA sequences. An “exon” is aportion of the sequence of a gene that is transcribed and is found inthe mature messenger RNA derived from the gene, and is not necessarily apart of the sequence that encodes the final gene product.

An “enhancing intron” is an intronic sequence present within thetranscribed region of a gene which is capable of enhancing expression ofthe gene when compared to an intronless version of an otherwiseidentical gene. An enhancing intronic sequence might also be able to actas an enhancer when located outside the transcribed region of a gene,and can act as a regulator of gene expression independent of position ororientation (Chan et. al. (1999) Proc. Natl. Acad. Sci. 96: 4627-4632;Flodby et al. (2007) Biochem. Biophys. Res. Commun. 356: 26-31).

The intron sequences can be operably linked to a promoter. Promoters maybe derived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven comprise synthetic DNA segments.

“Transcription terminator”, “termination sequences”, or “terminator”refer to DNA sequences located downstream of a coding sequence in agene, including polyadenylation recognition sequences and othersequences encoding regulatory signals capable of affecting mRNAprocessing or gene expression. The polyadenylation signal is usuallycharacterized by affecting the addition of polyadenylic acid tracts tothe 3′ end of the mRNA precursor. The use of different 3′ non-codingsequences is exemplified by Ingelbrecht, I. L., et al., Plant Cell1:671-680 (1989). A polynucleotide sequence with “terminator activity”refers to a polynucleotide sequence that, when operably linked to the 3′end of a second polynucleotide sequence that is to be expressed, iscapable of terminating transcription from the second polynucleotidesequence. Transcription termination is the process by which RNAsynthesis by RNA polymerase is stopped and both the RNA and the enzymeare released from the DNA template.

Improper termination of an RNA transcript can affect the stability ofthe RNA, and hence can affect protein expression. Variability oftransgene expression is sometimes attributed to variability oftermination efficiency (Bieri et al (2002) Molecular Breeding 10:107-117).

As used herein, a “heterologous terminator” is a terminator sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition by deliberatehuman intervention. A “heterologous terminator” is a polynucleotidesequence that exhibits “terminator activity”. Examples of suchmodifications from the native form to get a “heterologous terminator”include, but are not limited to, modifications by adding heterologoussequence elements to a terminator. A “heterologous terminator” as usedherein does not correspond to an endogenous terminator that is placed asa non-native chromosomal position.

The terms “SB-GKAF terminator”, “GKAF terminator” and “gamma-kafirinterminator” are used interchangeably herein, and each refers to thesequence encoding the 3′ untranslated region (3′ UTR) of the Sorghumbicolor gamma-kafirin gene and the about 300 bp region downstream fromthe 3′ UTR. The sequence of the SB-GKAF terminator is given in SEQ IDNO:1. Sorghum bicolor gamma-kafirin gene encodes a gamma-prolaminprotein, and the sequence for this gene is given in NCBI GI NO: 671655.Prolamins are the major storage proteins of many cereals. Thegamma-kafirin protein, which is the γ-prolamin of sorghum, constitutesabout 2-5% of total prolamin in sorghum endosperm, and is composed of asingle polypeptide of 27 kDa (de Freitas F A et al (1994) Mol Gen Genet245:177-186).

The terms “real-time PCR”, “quantitative PCR”, “quantitative real-timePCR” and “QPCR” are used interchangeably herein, and represent avariation of the standard polymerase chain reaction (PCR) technique usedto quantify DNA or RNA in a sample. Using sequence-specific primers anda probe, the relative number or copies of a particular DNA or RNAsequence are determined. The term relative is used since this techniquecompares relative copy numbers between different genes with respect to aspecific reference gene. The quantification arises by measuring theamount of amplified product at each cycle during the PCR process.Quantification of amplified product is obtained using fluorescenthydrolysis probes that measure increasing fluorescence for eachsubsequent PCR cycle. The “Ct” or “CT” (cycle threshold) is defined asthe number of cycles required for the fluorescent signal to cross thethreshold (i.e., exceeds background level). DNA/RNA from genes withhigher copy numbers will appear after fewer PCR cycles; so the lower aCt value, the more copies are present in the specific sample. Toquantify RNA, QPCR or real-time PCR is preceded by the step of reversetranscribing mRNA into cDNA. This is referred to herein as “real-timeRT-PCR” or “quantitative RT-PCR” or “qRT-PCR”.

The Taqman method of PCR product quantification uses a fluorescentreporter probe. This is more accurate since the probe is designed to besequence-specific and will only bind to the specific PCR product. Theprobe specificity allows for quantification even in the presence ofnon-specific DNA amplification. This allows for multiplexing, whichquantitates several genes in the same tube, by using probes withdifferent emission spectra. Breakdown of the probe by the 5′ to 3′exonuclease activity of Taq polymerase removes the quencher and allowsthe PCR product to be detected.

When plotted on a linear scale, the fluorescent emission increase withPCR cycle number has a sigmoidal shape with an exponential phase and aplateau phase. The plateau phase is determined by the amount of primerin the master mix rather than the nucleotide template. Usually thevertical scale is plotted in a logarithmic fashion, allowing theintersection of the plot with the threshold to be linear and more easilyvisualized. Theoretically, the amount of DNA doubles every cycle duringthe exponential phase, but this is affected by the efficiency of theprimers used. A positive control using a reference gene, e.g., a“housekeeping” gene that is relatively abundant in all cell types, isalso performed to allow for comparisons between samples. The amount ofDNA/RNA is determined by comparing the results to a standard curveproduced by serial dilutions of a known concentration of DNA/RNA.

As will be evident to one of skill in the art, any heterologouspolynucleotide of interest can be operably linked to the heterologousterminator sequence described in the current invention, and the methodsof the current invention can be used to assay the expression and copynumber of any heterologous polynucleotide of interest. Examples ofheterologous polynucleotides of interest that can be operably linked tothe heterologous terminator sequence and used for assaying copy numberusing the methods described in this invention include, but are notlimited to, heterologous polynucleotides comprising regulatory elementssuch as introns, enhancers, promoters, translation leader sequences,protein coding regions, or polynucleotides that can be used to controlgene expression. Examples of heterologous polynucleotides of interestthat can be operably linked to the heterologous terminator sequence andused for assaying gene expression using the methods described in thisinvention include, but are not limited to, regulatory elements such asintrons, protein coding polynucleotide sequences or polynucleotidesequences that control gene expression. Examples of protein-codingpolynucleotide sequences include, but are not limited to disease andinsect resistance genes, genes conferring nutritional value, genesconferring yield and heterosis increase, genes that confer male and/orfemale sterility, antifungal, antibacterial or antiviral genes, and thelike. Examples of heterologous polynucleotides that could be used tocontrol gene expression, include, but are not limited to, antisenseoligonucleotides, suppression DNA constructs, or nucleic acids encodingtranscription factors.

Using gene specific primer sets for quantization of transgene DNA/RNA intransgenic plants has drawbacks, as it can be costly when done in a highthroughput manner to have gene-specific primer for each differenttransgene, efficiency of each primer set might be different, which wouldhinder transgene copy number and expression assays in a high throughputfashion. Moreover gene-specific primer sets are not directly comparable,QPCR scores of two primer sets might not be equal. Moreover, expressionassays using gene-specific primer sets might not be transgene specific;e.g. if the transgene being tested is endogenous to the organism inwhich it is placed, a gene specific primer set will pick up bothtransgenic and endogenous expression.

One embodiment of this invention is the use of the method disclosedherein for quantifying expression or copy number of a heterologouspolynucleotide in more than one transgenic plant, wherein eachtransgenic plant comprises a recombinant construct comprising adifferent heterologous polynucleotide operably linked to a heterologousterminator comprising the same polynucleotide sequence. In oneembodiment the method disclosed herein is used for quantifyingexpression and copy number of a heterologous polynucleotide in manytransgenic plants, in a high-throughput manner, wherein each transgenicplant comprises a recombinant construct comprising a differentheterologous polynucleotide operably linked to a heterologous terminatorcomprising the same polynucleotide sequence. In one embodiment theheterologous terminator is the SB-KAF terminator. In one embodiment, thetransgenic plants are maize plants.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal V method of alignment(Higgins and Sharp, CABIOS. 5:151-153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments and calculation of percent identity of proteinsequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters areKTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignmentof the sequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Alternatively, the Clustal W method of alignment may be used. TheClustal W method of alignment (described by Higgins and Sharp, CABIOS.5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191(1992)) can be found in the MegAlign™ v6.1 program of the LASERGENE®bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Defaultparameters for multiple alignment correspond to GAP PENALTY=10, GAPLENGTH PENALTY=0.2, Delay Divergent Sequences=30%, DNA TransitionWeight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB.For pairwise alignments the default parameters areAlignment=Slow-Accurate, Gap Penalty=10.0, Gap Length=0.10, ProteinWeight Matrix=Gonnet 250 and DNA Weight Matrix=IUB. After alignment ofthe sequences using the Clustal W program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table in the same program.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press Cold Spring Harbor, 1989(hereinafter “Sambrook”). Embodiments of the current invention include:

One embodiment of this invention is a method of quantifying the level ofexpression of a heterologous polynucleotide in a transgenic plant orplant cell, the method comprising the steps of: (a) isolating nucleicacids from a transgenic plant or plant cell, wherein the transgenicplant or plant cell comprises a heterologous polynucleotide operablylinked to a heterologous terminator sequence; and (b) quantifying thelevel of expression of the heterologous polynucleotide by real-timereverse transcriptase polymerase chain reaction using a forward primerand a reverse primer, wherein the forward primer and the reverse primerhybridize to the heterologous terminator sequence or the complementthereof. In another embodiment, the quantification of level ofexpression of the heterologous polynucleotide is done by quantitativereverse transcriptase real-time PCR using a probe that hybridizes to theheterologous terminator sequence or the complement thereof.

Another embodiment of the present invention is a method of measuring thecopy number of a heterologous polynucleotide in a transgenic plant orplant cell, the method comprising the steps of: (a) isolating nucleicacids from a transgenic plant or plant cell, wherein the transgenicplant or plant cell comprises a heterologous polynucleotide operablylinked to a heterologous terminator sequence; and (b) quantifying thecopy number of the heterologous polynucleotide by real-time polymerasechain reaction using a forward primer and a reverse primer, wherein theforward primer and the reverse primer hybridize to the heterologousterminator sequence or the complement thereof. In another embodiment,the quantification of copy number of the heterologous polynucleotide isdone by quantitative real-time PCR using a probe that hybridizes to theheterologous terminator sequence or the complement thereof.

Another embodiment of the invention is a method of quantifying the levelof expression of at least two heterologous polynucleotides present in atleast two transgenic plants or plant cells, the method comprising thesteps of: (a) isolating nucleic acids from at least two transgenicplants or plant cells, wherein a first transgenic plant or plant cellcomprises a first heterologous polynucleotide operably linked to aheterologous terminator sequence, and wherein a second transgenic plantor plant cell comprises a second heterologous polynucleotide operablylinked to the heterologous terminator sequence; (b) optionally,isolating nucleic acids from additional transgenic plants or plantcells, wherein each of the additional transgenic plants or plant cellscomprises an additional heterologous polynucleotide operably linked tothe heterologous terminator sequence; and (c) quantifying the level ofexpression of the first heterologous polynucleotide, the secondheterologous polynucleotide and optional additional heterologouspolynucleotides by real-time reverse transcriptase polymerase chainreaction using a forward primer and a reverse primer, wherein theforward primer and the reverse primer hybridize to the heterologousterminator sequence or the complement thereof. In one embodiment, theisolation of nucleic acids from additional transgenic plants or plantcells of step (b) and quantification of level of expression of optionaladditional heterologous polynucleotides of step (c) is done for at least100, at least 200, at least 300, at least 400, at least 500, at least600, at least 700, at least 800, at least 900, or at least 1000additional transgenic plants or plant cells. In one embodiment the copynumber of the at least two heterologous polynucleotides present in theat least two transgenic plants or plant cells, wherein each heterologouspolynucleotide is operably linked to the heterologous terminator, isquantified using this method.

In another embodiment of any of the methods described herein, theheterologous terminator sequence comprises a SB-GKAF terminatorsequence. In another embodiment, the sequence of the heterologousterminator sequence comprises SEQ ID NO:1. In another embodiment, theforward primer, the reverse primer and the probe hybridize to SEQ IDNO:1 or the complement thereof. In another embodiment, the heterologousterminator sequence comprises the SB-GKAF terminator sequence, and theprobe hybridizes to the region of the SB-GKAF terminator sequencebounded by the forward primer and the reverse primer. In anotherembodiment, the heterologous terminator sequence comprises SEQ ID NO: 1,the forward primer comprises SEQ ID NO:2, the reverse primer comprisesSEQ ID NO:3 and the probe comprises SEQ ID NO:4. In another embodiment,the heterologous terminator sequence comprises SEQ ID NO:1, the forwardprimer comprises SEQ ID NO:5, the reverse primer comprises SEQ ID NO:6and the probe comprises SEQ ID NO:7. In another embodiment, theheterologous terminator sequence comprises SEQ ID NO:1, the forwardprimer comprises SEQ ID NO:2, the reverse primer comprises SEQ ID NO:6and the probe comprises at least one sequence selected from the groupconsisting of SEQ ID NO:4 and SEQ ID NO:7. In another embodiment, any ofthe methods described herein, wherein the plant or plant cell is amonocotyledonous or dicotyledonous plant or plant cell, for example, amaize or soybean plant or plant cell. The plant or plant cell may alsobe from sunflower, sorghum, canola, wheat, alfalfa, cotton, rice,barley, millet, sugar cane or switchgrass. The invention encompassesregenerated, mature and fertile transgenic plants generated using themethods described above, transgenic seeds produced therefrom, T1 andsubsequent generations.

EXAMPLES

The present invention is further illustrated in the following Examples,in which parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these examples,while indicating embodiments of the invention, are given by way ofillustration only. From the above discussion and these Examples, oneskilled in the art can ascertain the essential characteristics of thisinvention, and without departing from the spirit and scope thereof, canmake various changes and modifications of the invention to adapt it tovarious usages and conditions. Furthermore, various modifications of theinvention in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Assaying Transgene Expression in Maize Using Sequence from aSB-GKAF Terminator

The sequence of the SB-GKAF terminator (gamma-kafirin terminator fromSorghum bicolor) is given in SEQ ID NO:1. Like the majority of all geneterminators, the SB-GKAF terminator is known to be affected bypost-transcriptional mRNA processing. In order to produce a reliableSB-GKAF terminator-specific QPCR (Quantitative Polymerase Chain Reactionor “real time PCR”) primer set, it was required to determine where theterminator would be processed and a poly-A tail would be added bypost-transcriptional processing. Without the knowledge of this location,there would have been the risk of creating a QPCR based assay that ispre-transcriptional processing mRNA specific, missing allpost-transcriptional mRNA. By finding and comparing the sequence ofSB-GKAF terminator given in SEQ ID NO:1 to sequences of other verysimilar SB-GKAF EST clones in Pioneer's internal data set, theprediction about where the SB-GKAF terminator would be clipped could bemade. This clipping site was then further confirmed by identifying thepolyA recognition sequence. The “safe zone” sequence, upstream of thepredicted terminator clipping site, was used to design the TaqManprimer/probe set. This primer/probe set was named SBTerm2.

Shown below is the sequence of the SB-GKAF terminator (SEQ ID NO:1). Thebinding sites for the SBTerm2 forward and reverse primers (SEQ ID NO:2and SEQ ID NO:3, respectively) are depicted in bold capital letters inthe sequence below. The SBTerm2 probe (SEQ ID NO:4) binding site isshown in italics and bold letters. The poly (A) recognition sequence isshown in lower case and bold. The sequence predicted to be clipped offduring mRNA processing is shown in italics.

SB-GKAF terminator (SEQ ID NO: 1):actaactatctatactgtaataatgttgtataGCCGCCGGA TAGCTAGCTag

gggtaat aataaagtgtcATCCATCCATCACCATGGGTggcaacgtgagcaatgacctgattgaacaaattgaaatgaaaagaagaaatatgttatatgtcaacgagatttcctcataatgccactgacgacgtgtgtccaagaaatgtatcagtgatacgtatattcacaatttttttatgacttatactcacaatttgtttttttactacttatactcacaatttgttgtgggtaccataacaatttcgatcgaatatatatcagaaagttgacgaaagtaagctcactcaaaaagttaaatgggctgcggaagctgcgtcaggcccaagttttggctattctatccggtatccacgattttgatggctgaggg acatatgttcgctt

Primer Design and Validation:

The SBTerm2 primer/probe set is Taqman based. For testing, the probe waslabeled with a VIC dye, but other dyes also can be used. Sequences ofthe SBTerm2 primer/probe are shown below (Table 1).

TABLE 1 SBTerm2 Primer and Probe Names, Sequences,  and Dye InformationPrimer/Probe Sequence Dye SBTerm2F GCCGCCGGATAGCTAGCT — (SEQ ID NO: 2)SBTerm2R ACCCATGGTGATGGATGGAT — (SEQ ID NO: 3) SBTerm2 ProbeTTTAGTCATTCAGCGGCGAT VIC (SEQ ID NO: 4)

The first step in validating the SBTerm2 primer/probe QPCR assay was bytesting the efficiency of the primer/probe set. To do this, a plasmidcontaining the SB-GKAF terminator was serial diluted, and the dilutionswere used as PCR templates for the SBTerm2 primer/probe set.

TABLE 2 The Slope and Efficiency Results of Primer/Probe Set SBTerm2Primer set Template Slope Efficiency SBTerm2 PHP31801 −3.580 0.903

The efficiency test proved to be successful (FIG. 1). The amplificationsfor primer/probe set SBTerm2 were clearly discernable (FIG. 2), and theefficiency was found to be about 90% (Table 2).

Gene of Interest (GOI)-Specific Vs. SB-GKAF Terminator-Specific PrimerSets:

Although a terminator is considered part of a gene, “gene-specific” or“GOI-specific” primer sets, used interchangeably herein, refer to primersets that are in the portion of the gene upstream of the terminator. Itis standard practice to assay gene expression by designing uniquegene-specific primer sets. For high throughput assays of transgeneexpression in transgenic maize plants, this can amount to literallythousands of genes, each with its own unique primer set. This method ofassaying gene expression is costly and has many drawbacks. The drawbacksof using gene-specific primer sets include cost, since each unique GOIrequired its own primer set. Additionally, there were efficiencyvariations since GOI-specific primer sets were not directly comparable;the QPCR score of one primer set might not equal the QPCR score ofanother primer set. Moreover, a gene-specific primer set is notnecessarily transgene specific; e.g., if the transgene being tested isendogenous to the organism in which it is placed, a gene-specific primerset will pick up both transgenic and endogenous expression.

If each of the Genes of Interest (GOIs) in a number of transgenic maizelines had a universal sequence transcribed, and if that sequence wasunique with respect to each transformed plant's genome, then just oneprimer set could be designed. This approach would solve the drawbacks ofhaving to use gene-specific primer sets. Furthermore, a vast majority oftransgenic GOIs are ESTs. ESTs come from mRNA transcripts whichnaturally do not contain full terminator sequences. Because ESTs aremissing a full terminator, when ESTs are used as a transgenic gene aterminator must be supplied for reliable transcription termination.

Materials and Methods: Sample Collection and Preparation:

All of the QPCRs mentioned below were done using transgenic Gaspe Flintderived maize lines containing GOIs. The transgenic Gaspe Flint derivedmaize lines containing GOIs may or may not have been challenged withabiotic stresses. All of the samples were taken from corn and werecollected by leaf punching followed by immediate sample freezing usingdry ice. All transgenic samples contain an over-expressed GOI that usesthe SB-GKAF terminator for transcription termination. All transgenes inthis study were native to corn. Because the GOIs were all native tocorn, after transformation, each transgenic plant would have at leasttwo copies of the GOI (the endogenous and the transgenic), and eachnon-transgenic plant only has one copy of the GOI (the endogenous).

From each sample, the mRNA was extracted, the genomic DNA was degraded,and cDNA was synthesized. From the cDNA, two QPCRs were done: a SB-GKAFterminator specific assay (SBTerm2), and an inner GOI specific assay(FIG. 3).

Correlation Analysis:

After the GOI and SB-GKAF terminator specific primer set QPCRs weredone, a construct specific correlation comparison was conducted. Thecorrelation comparison was done to illustrate that a SB-GKAF terminatorspecific primer set can be used to detect transgenic expression, bycomparison to the standard GOI specific QPCR method. The reasoning ofthe correlation analysis was that because the two PCRs are specific forthe same transcription fragment (FIG. 3), any changes in expression ofthe GOI specific QPCR should also have corresponding changes in theSB-GKAF terminator specific QPCR.

Endogenous GOI Expression Interference:

In this study, the correlation comparison of the two primer sets can bethrown off if a sample is included in the analysis that does not havetheir transgenic GOI expressing at a level higher than the endogenousgene. As stated above, all of the transgenic GOIs in this study arenative to the transformed organism. This can throw off the correlationanalysis because even though the SBTerm2 primer set is specific for theSB-GKAF terminator, the inner GOI primer set not only detects transgenicexpression (as shown in FIG. 3), but also detects endogenous GOIexpression (not shown). This would not be a problem if the transgenicGOIs were completely foreign in sequence, since then, like with theSB-GKAF terminator primer set, the inner GOI specific primer set wouldbe specific to the transgene.

To bypass this error, the upper limit of endogenous expression wasdetected and only the samples expressing their transgene beyond thispoint were focused on. The idea was that even if the endogenous geneexpression were changing (which could throw off correlations), it wouldhave made little or no difference if the transgenic GOI was expressingat a much higher level and only those much higher levels were used inthe correlation analysis. The endogenous GOI expression zone was foundby identifying the lowest inner GOI primer set CT scores ofnon-transgenics or transgenic non-expressors, if available. This lowestCT value was then used to set a threshold CT score, and any transgenicsamples that had a higher inner GOI CT scores (lower expression) thanthis threshold were not used in the correlation analysis, because it wasdeemed to not be expressing the transgene beyond the “endogenous zone”.

In addition to transgenic samples, QPCRs were also done onnon-transgenic samples, or endogenous expression levels were deduced bysearching for transgenic samples not expressing the transgene, as foundby not expressing the SB-GKAF terminator specific primer set. If neitherof these two options for finding endogenous GOI expression level wereavailable, all of that construct's transgenic samples were used in theinner GOI primer set vs. SB-GKAF primer set correlation comparison.

Results:

Thirty-three transgenic maize lines with unique constructs were testedfor transgene expression using GOI specific primers and SB-GKAFterminator specific primers. Multiple events were tested for eachconstruct. Table 3 depicts the calculation of the SBTerm2-GOI CTCorrelation Coefficient data for one construct. Table 4 depicts theSBTerm2-GOI CT Correlation Coefficient for the 33 constructs tested andaverage of the SBTerm2-GOI CT Correlation Coefficient.

Table Heading Explanations:

-   -   1. Sample—Each sample represents a unique transformation event        or a unique non-transgenic control plant.    -   2. Construct—Each construct refers to a unique vector. Each        vector is identical, except for the GOI that is being        over-expressed. Constructs labeled “NT” are non-transgenic, and        were not transformed with a vector.    -   3. Inner GOI Primer set—Each construct had a unique GOI, and        each GOI has a unique inner gene primer set. Each transgenic GOI        comes from an EST. After vector insertion, each EST uses the        SB-GKAF terminator. Non-transgenic controls do not have        transgenic GOIs, but like the transgenic samples, do have the        endogenous GOI.    -   4. SBTerm2 CT—The QPCR CT score for the SB-GKAF terminator        specific primer set SBTerm2.    -   5. Inner GOI Primer Set CT—The QPCR CT score of whatever inner        GOI primer set was run for a particular sample.    -   6. Used in Correlation—States whether or not a sample's SBTerm2        and inner GOI CT scores were used in the correlation analysis.        Along with non-transgenic samples, samples showing GOI        expression at endogenous levels were not included in the        correlation analysis.    -   7. GOI CT Cutoff—The cutoff inner GOI primer set CT score used        as a threshold. This threshold is the highest level of        endogenous expression (lowest CT score) found in non-transgenic        samples or transgenic samples not expressing transgenic GOI (as        found by not expressing SBTerm2). Any sample that does not have        an inner GOI primer set CT score below the threshold CT is        considered to not be expressing their transgenic GOI higher than        natural levels, and thus are not to be included in the        correlation analysis.    -   8. SBTerm2-GOI CT Correlation Coefficient—The correlation        coefficient found using the specified transgenic samples        included in analysis.

TABLE 3 CT and SBTerm2-GOI CT Correlation Coefficient Data for aSpecific Transgenic Construct Inner Inner GOI GOI Primer Used in Con-Primer SBTerm2 Set Correla- Sample struct Set CT CT tion? Transgenic 1TR004 25.487 20.586 Yes event 1 Transgenic 1 TR004 25.151 20.701 Yesevent 2 Transgenic 1 TR004 25.066 20.875 Yes event 3 Transgenic 1 TR00425.474 20.919 Yes event 4 Transgenic 1 TR004 25.984 21.461 Yes event 5Transgenic 1 TR004 24.954 21.572 Yes event 6 Transgenic 1 TR004 26.12921.834 Yes event 7 Transgenic 1 TR004 26.898 21.957 Yes event 8Transgenic 1 TR004 26.460 22.196 Yes event 9 Transgenic 1 TR004 26.53222.237 Yes event 10 Transgenic 1 TR004 26.372 22.341 Yes event 11Transgenic 1 TR004 26.902 22.354 Yes event 12 Transgenic 1 TR004 28.31523.054 Yes event 13 Transgenic 1 TR004 27.286 23.090 Yes event 14Transgenic 1 TR004 29.663 24.790 Yes event 15 GOI CT 30.00 cut offSBTerm2-  0.939 GOI CT Correlation Coefficient

TABLE 4 Summary and Average of SBTerm2-GOI CT Correlation CoefficientData for 33 Transgenic Constructs SBTerm2-GOI CT Primer CorrelationConstruct set Coefficient 1 TR004 0.939 2 TR005 0.981 3 TR038 0.936 4TR032 0.983 5 TR034 0.813 6 TR026 0.994 7 TR023 0.902 8 TR064 0.910 9TR053 0.874 10 TR055 0.878 11 TR008 0.880 12 TR010 0.969 13 TR072 0.98114 TR018 0.914 15 TR002 0.899 16 TR069 0.968 17 TR079 0.873 18 TR0950.865 19 TR084 0.757 20 TR101 0.734 21 TR093 0.838 22 TR100 0.720 23TR086 0.973 24 TR108 0.917 25 TR104 0.932 26 TR083 0.919 27 TR082 0.84228 TR109 0.816 29 TR110 0.917 30 TR122 0.891 31 TR123 0.997 32 TR1260.410 33 TR127 0.819 Ave 0.88

A distinct advantage of the SB-Term terminator primer set QPCR assay isthat it is transgene specific. The ability to decipher transgenicexpressers from transgenic non-expressors provides a tremendousadvantage when compared to the use of only inner GOI primer sets.

The level of endogenous GOI expression levels could be inferred from 21of the constructs that had non-transgenic controls included, and 3constructs that contained transgenic samples that were not expressingthe transgene (as was evident by having SBTerm2 CT scores in the 30s).For these 24 constructs, a threshold value could be adequately made toexclude any transgenic samples that were not expressing the transgene athigher than endogenous levels. The remaining 10 constructs, in whichendogenous expression levels could not be inferred, had all samplesincluded in their construct specific correlation analysis.

As is evident from the correlation coefficients, it appears that theexpression variations of the inner GOI specific primer sets and theSBTerm2 primer set change in a correlated manner. These high valuepositive correlations showed that both primer sets are monitoring theexpression of the same transcripts. There were 17 constructs that had acorrelation above 90%, 13 constructs between 80-90%, 3 constructsbetween 70-80%, and one construct below 70%. These are high correlationvalues, considering the error of endogenous expression interference andprimer set efficiency variation. The correlation coefficients with themost weight might be constructs that had little or no endogenous GOIexpression, since they would be likely to have the least amount ofendogenous GOI expression interference. The correlation coefficientvalues of these select constructs were all 80% or above.

While all of the constructs in these experiments were over-expressingthe GOI, the SB-GKAF terminator can also be used in RNAi knockdownconstructs. However, one would not expect correlated values betweeninner GOI primer sets and SB-GKAF terminator specific primer sets,because the knock down machinery would be playing too large of aninterfering role on mRNA transcripts.

Example 2 Alternative Primer/Probe Set for Assaying Transgene Expressionin Maize

Another primer/probe set, designated SBTerm1, was used to assaytransgene expression in maize. The SBTerm1 and SBTerm2 primer/probe setswere found to have similar amplification efficiencies. Sequences of theSBTerm1 primer/probe set are shown below (Table 5).

TABLE 5 SBTerm1 Primer and Probe Names and Corresponding SequencesPrimer/Probe Sequence SBTerm1F GGCGATGGGTAATAATAAAGTGTCA (SEQ ID NO: 5)SBTerm1R CAATCAGGTCATTGCTCACGTT (SEQ ID NO: 6)  SBTerm1 ProbeCATCCATCACCATGGGT (SEQ ID NO: 7)

Example 3 Assaying Copy Number in Maize Using Sequence from a SB-GKAFTerminator

Primers and probe sequence specific to the SB-GKAF terminator sequencecan also used to determine copy number of a transgene with QPCR in ahigh-throughput assay.

For zygosity or copy number assays, a known fully segregated homozygoushousekeeping gene that has two copies can be compared to unknown samplesthat are either homozygous, heterozygous or have no copies of thetransgene tested. The relative comparison of the samples would determinethe copy number present for the unknown sample.

Reactions can be carried out for 40 PCR cycles on an ABI Taqman 7900 PCRinstrument, with cycling parameters of 95° C. for 2 min, 95° C. for 10sec, 60° C. for 1 min. Fluorescent measurements can be taken from eachwell at each of the 40 cycles for both the terminator sequence derivedfrom the Sorghum bicolor GKAF (SB-GKAF) gene and the endogenous adh1control. Samples can be scored for relative copy number by subtractingthe cycle threshold values from the cycle threshold value of theendogenous control. The cycle threshold (Ct) can be determined, and thedelta Ct can be calculated relative to the known endogenous controlvalue.

Example 4 Assaying GUS Transgene Expression in Maize Using Sequence froma SB-GKAF Terminator

An SB-GKAF terminator expression assay was also done on transgenic maizeplants transformed with constructs containing a GUS protein-codingsequence operably linked to an SB-GKAF terminator. GUS is not endogenousto corn. The number of samples assayed was 108, which included twotissues (root and leaf) and seven Promoter::GUS::SB-GKAF constructs. Anumber of different promoters were used to drive GUS expression. Copynumber analysis indicated that for each transgenic line the T-DNA was asingle, non-complex insert. For each sample, we ran QPCR primer sets onthe following three elements: a reference gene (eIF4g), GUS, and theSB-GKAF terminator. The sequences of the eIF4g and GUS primers sets andprobes are given in Table 6. The sequences primers and probe used forSB-GKAF are given in Table 1 (SBTerm2F, SBTerm2R and SBTerm2 probe; SEQID NO:2, 3 and 4 respectively). The results are given in Table 7 and inFIG. 4. FIG. 4 shows a QPCR CT comparison using GUS and SB-GKAFterminator primer sets run on the same transgenic samples. GUS CTs areon the Y axis and SB-GKAF CTs are on the X axis. The correlationcoefficient for this is 0.9805 with an R̂2 of 0.961. As is evident fromthe correlation coefficient, expression levels based on the GUS-specificprimer set and the SBTerm2 primer set are correlated. This high positivecorrelation value shows that either primer sets can be used to determineexpression levels of the GUS transgene.

TABLE 6 eIF4g and GUS Primer and Probe Names, Sequences, and Dye Information Primer/Probe Sequence Dye GUS-1482-FCGGAAGCAACGCGTAAACTC — (SEQ ID NO: 8) GUS-1553-R TGTGAGCGTCGCAGAACATTA —(SEQ ID NO: 9) GUS-1509-P CGCGTCCGATCACCTGCGTC FAM (SEQ ID NO: 10)elF4-g-F CCTCCTCGAGCCATTTGACA — (SEQ ID NO: 11) elF4-g-RAGGGCAGGCAATCTTTCGT — (SEQ ID NO: 12) elF4-g-P ACGGCTCCAGAGCT VIC(SEQ ID NO: 13)

TABLE 7 Tissue Ct-Reference CT- CT- type Sample name PHP (eIF4g) GUSSBGKAF Leaf AP0140D01L.16 49798 21.92 20.57 20.84 Leaf AP0140F01L.1649803 20.87 20.78 20.71 Root AP0140F01R.8 49803 19.57 20.89 21.45 LeafAP0140F01L.9 49803 21.61 20.92 21.81 Root AP0140F01R.16 49803 19.5921.02 22.28 Leaf AP0140D01L.1 49798 22.05 21.19 20.9 Root AP0140F01R.1049803 19.64 21.45 21.97 Leaf AP0140F01L.12 49803 23.63 21.5 21.7 LeafAP0140D01L.8 49798 21.65 21.86 22.34 Root AP0140F01R.7 49803 19.75 21.9222.32 Root AP0140F01R.6 49803 20.26 22.07 22.83 Leaf AP0140F01L.7 4980320.66 22.17 22.68 Leaf AP0140B01L.9 49794 22.72 22.42 22.46 RootAP0140F01R.9 49803 20.64 22.69 23.96 Leaf AP0140F01L.8 49803 21.91 22.7422.73 Leaf AP0140D01L.15 49798 22.42 22.85 23.48 Root AP0140C01R.8 4979620.02 22.87 23.77 Leaf AP0140F01L.1 49803 21.73 22.88 22.96 LeafAP0140F01L.2 49803 21.7 22.92 22.74 Root AP0140F01R.14 49803 20.46 22.9622.97 Leaf AP0140F01L.5 49803 23.51 23.23 23.5 Root AP0140F01R.1 4980320.23 23.24 24.13 Root AP0140F01R.15 49803 20.12 23.27 23.75 RootAP0140F01R.4 49803 21.06 23.61 24.14 Root AP0140F01R.13 49803 20.8323.61 23.92 Leaf AP0140F01L.14 49803 23.54 23.83 23.67 RootAP0140F01R.11 49803 21.3 23.9 24.71 Root AP0140C01R.9 49796 20.19 23.9124.78 Leaf AP0140F01L.6 49803 22.84 24.08 24.47 Leaf AP0140C01L.9 4979622.99 24.18 25.45 Leaf AP0140F01L.15 49803 22.83 24.31 24.39 LeafAP0140D01L.10 49798 22.84 24.5 24.84 Leaf AP0140D01L.14 49798 24.1124.51 24.34 Leaf AP0140C01L.8 49796 21.87 24.57 25.16 Leaf AP0140F01L.449803 23.68 24.62 25.06 Root AP0140F01R.5 49803 20.82 24.71 24.84 LeafAP0140F01L.3 49803 23.74 24.81 24.75 Leaf AP0140D01L.6 49798 23.99 24.8824.8 Root AP0140G01R.10 49802 21.28 24.93 25.69 Root AP0140C01R.7 4979621.91 25.15 26.06 Root AP0140C01R.16 49796 20.67 25.18 26.31 LeafAP0140C01L.1 49796 22.01 25.36 26.77 Root AP0140F01R.12 49803 22.3 25.3625.04 Leaf AP0140F01L.13 49803 23.81 25.45 25.89 Leaf AP0140C01L.1549796 22.76 25.46 26.3 Root AP0140G01R.7 49802 20.12 25.56 27.42 LeafAP0140D01L.7 49798 23.48 25.63 25.98 Root AP0140C01R.15 49796 20.6525.66 26.65 Leaf AP0140C01L.16 49796 22.04 25.67 26.43 Root AP0140C01R.149796 20.86 25.69 26.74 Leaf AP0140C01L.4 49796 23.76 25.84 25.92 RootAP0140G01R.13 49802 19.96 25.92 25.81 Leaf AP0140C01L.7 49796 23.3425.93 26.78 Root AP0140B01R9 49794 21.02 26.11 26.53 Root AP0140G01R.1449802 20.29 26.32 27.1 Root AP0140G01R.16 49802 19.8 26.38 27.53 RootAP0140G01R.2 49802 21.88 26.4 27.52 Leaf AP0140C01L.2 49796 22.58 26.5327.45 Root AP0140C01R.10 49796 21.28 26.62 28.2 Root AP0140C01R.6 4979621.39 26.64 28.63 Root AP0140G01R.11 49802 20.33 26.81 27.07 RootAP0140C01R.2 49796 22.18 26.86 28.32 Root AP0140G01R.6 49802 20.03 27.0428.21 Leaf AP0140D01L.12 49798 25.53 27.12 27.3 Leaf AP0140D01L.4 4979824.37 27.21 27.88 Root AP0140G01R.8 49802 20.02 27.25 28.7 LeafAP0140C01L.12 49796 24.19 27.32 27.6 Leaf AP0140A01L.3 49792 24.57 27.4228.22 Leaf AP0140C01L.10 49796 23.26 27.43 28.36 Leaf AP0140C01L.3 4979624.04 27.46 27.78 Root AP0140C01R.12 49796 24.23 27.47 28.67 LeafAP0140C01L.6 49796 23.57 27.53 27.94 Root AP0140C01R.14 49796 23.2 27.5729.91 Leaf AP0140C01L.14 49796 23.06 27.62 27.99 Leaf AP0140D01L.3 4979823.88 27.65 27.69 Root AP0140C01R.4 49796 23.22 27.65 29.52 LeafAP0140C01L.5 49796 23.77 27.69 28.54 Leaf AP0140C01L.11 49796 23.9427.69 27.9 Leaf AP0140C01L.13 49796 23.97 27.72 28.36 Root AP0140G01R.949802 20.66 27.74 28.81 Root AP0140G01R.15 49802 21.75 27.88 29.65 LeafAP0140A01L.15 49792 21.77 27.92 28.96 Root AP0140C01R.11 49796 23.5728.03 29.9 Root AP0140C01R.13 49796 23.71 28.09 29.63 Leaf AP0140G01L.1149802 22.86 28.23 28.5 Root AP0140C01R.5 49796 23.78 28.3 30.44 LeafAP0140A01L.2 49792 23.14 28.5 29.35 Root AP0140A01R.15 49792 20.37 28.5329.25 Root AP0140A01R.7 49792 21.67 28.63 28.97 Root AP0140D01R.2 4979821.53 28.69 30.26 Leaf AP0140A01L.8 49792 22.51 28.75 27.36 LeafAP0140D01L.2 49798 23.61 28.76 29.87 Root AP0140G01R.4 49802 21.62 28.8929.57 Leaf AP0140A01L.11 49792 24.49 29.23 29.84 Root AP0140G01R.3 4980221.62 29.27 29.74 Root AP0140G01R.12 49802 23.15 29.32 29.97 LeafAP0140E01L.1 49801 22.28 29.32 30.75 Leaf AP0140G01L.1 49802 22.94 29.3330.38 Leaf AP0140A01L.7 49792 22.91 29.37 29.87 Leaf AP0140E01L.11 4980124.65 29.53 30.7 Root AP0140C01R.3 49796 25.11 29.71 30.98 LeafAP0140A01L.6 49792 23.33 29.82 30.81 Leaf AP0140A01L.5 49792 24.08 29.8731.5 Leaf AP0140A01L.9 49792 22.88 29.88 31.07 Leaf AP0140A01L.14 4979223.92 29.89 31.05 Root AP0140A01R.10 49792 21.64 29.92 31.59 RootAP0140A01R.3 49792 21.36 29.93 30.5 Root AP0140A01R.9 49792 20.74 29.9731.25

1. A method of quantifying the level of expression of a heterologouspolynucleotide in a transgenic plant or plant cell, the methodcomprising the steps of: a. isolating nucleic acids from a transgenicplant or plant cell, wherein the transgenic plant or plant cellcomprises a heterologous polynucleotide operably linked to aheterologous terminator sequence; and b. quantifying the level ofexpression of the heterologous polynucleotide by real-time reversetranscriptase polymerase chain reaction using a forward primer and areverse primer, wherein the forward primer and the reverse primerhybridize to the heterologous terminator sequence or the complementthereof.
 2. A method of measuring the copy number of a heterologouspolynucleotide in a transgenic plant or plant cell, the methodcomprising the steps of: a. isolating nucleic acids from a transgenicplant or plant cell, wherein the transgenic plant or plant cellcomprises a heterologous polynucleotide operably linked to aheterologous terminator sequence; and b. quantifying the copy number ofthe heterologous polynucleotide by real-time polymerase chain reactionusing a forward primer and a reverse primer, wherein the forward primerand the reverse primer hybridize to the heterologous terminator sequenceor the complement thereof.
 3. The method of claim 1, wherein theheterologous terminator sequence is a SB-GKAF terminator sequence. 4.The method of claim 2, wherein the heterologous terminator sequence is aSB-GKAF terminator sequence.
 5. (canceled)
 6. (canceled)
 7. (canceled)8. The method of claim 1 wherein the quantification of the level ofexpression of the heterologous polynucleotide of step (b) is done byreal-time reverse transcriptase PCR using a probe that hybridizes to theheterologous terminator sequence or the complement thereof.
 9. Themethod of claim 2 wherein the quantification of the copy number of theheterologous polynucleotide of step (b) is done by real-time PCR using aprobe that hybridizes to the heterologous terminator sequence or thecomplement thereof.
 10. The method of claim 8, wherein the heterologousterminator comprises a SB-GKAF terminator sequence and the probehybridizes to the region of the SB-GKAF terminator sequence bounded bythe forward primer and the reverse primer.
 11. The method of claim 10,wherein the forward primer comprises SEQ ID NO:2, the reverse primercomprises SEQ ID NO:3 and the probe comprises SEQ ID NO:4.
 12. Themethod of claim 10, wherein the forward primer comprises SEQ ID NO:5,the reverse primer comprises SEQ ID NO:6 and the probe comprises SEQ IDNO:7.
 13. The method of claim 8, wherein the transgenic plant or plantcell is a maize plant or plant cell.
 14. A method of quantifying thelevel of expression of at least two heterologous polynucleotides presentin at least two transgenic plants or plant cells, the method comprisingthe steps of: a. isolating nucleic acids from at least two transgenicplants or plant cells, wherein a first transgenic plant or plant cellcomprises a first heterologous polynucleotide operably linked to aheterologous terminator sequence, and wherein a second transgenic plantor plant cell comprises a second heterologous polynucleotide operablylinked to the heterologous terminator sequence; b. optionally, isolatingnucleic acids from additional transgenic plants or plant cells, whereineach of the additional transgenic plants or plant cells comprises anadditional heterologous polynucleotide operably linked to theheterologous terminator sequence; and c. quantifying the level ofexpression of the first heterologous polynucleotide, the secondheterologous polynucleotide and the optional additional heterologouspolynucleotides by real-time reverse transcriptase polymerase chainreaction using a forward primer and a reverse primer, wherein theforward primer and the reverse primer hybridize to the heterologousterminator sequence or the complement thereof.
 15. The method of claim14 wherein the steps of isolation of nucleic acids and quantifying thelevel of expression of heterologous polynucleotides from additionaltransgenic plants are done for at least one hundred additionaltransgenic plants or plant cells.
 16. The method of claim 14, whereinthe heterologous terminator comprises a SB-GKAF terminator sequence andthe quantifying step uses a probe which hybridizes to the region of theSB-GKAF terminator sequence bounded by the forward primer and thereverse primer.
 17. The method of claim 16, wherein the forward primercomprises SEQ ID NO:2, the reverse primer comprises SEQ ID NO:3 and theprobe comprises SEQ ID NO:4.
 18. The method of claim 16, wherein theforward primer comprises SEQ ID NO:5, the reverse primer comprises SEQID NO:6 and the probe comprises SEQ ID NO:7.
 19. The method of claim 14,wherein the transgenic plant or plant cell is a maize plant or plantcell.
 20. The method of claim 15, wherein the transgenic plant or plantcell is a maize plant or plant cell.
 21. The method of claim 9, whereinthe heterologous terminator comprises a SB-GKAF terminator sequence andthe probe hybridizes to the region of the SB-GKAF terminator sequencebounded by the forward primer and the reverse primer.
 22. The method ofclaim 21, wherein the forward primer comprises SEQ ID NO:2, the reverseprimer comprises SEQ ID NO:3 and the probe comprises SEQ ID NO:4. 23.The method of claim 21, wherein the forward primer comprises SEQ IDNO:5, the reverse primer comprises SEQ ID NO:6 and the probe comprisesSEQ ID NO:7.
 24. The method of claim 9, wherein the transgenic plant orplant cell is a maize plant or plant cell.